HA标签的水稻水孔蛋白<i>OsPIP2;7</i>基因植物双元表达载体构建及转基因水稻的获得

doi:10.7668/hbnxb.201750580

摘要/Abstract

摘要： 为研究水稻水孔蛋白OsPIP2；7基因在非生物胁迫中的生物学功能，根据OsPIP2；7基因序列设计特异性引物，以水稻品种中花11的三叶龄叶片RNA反转录的cDNA第一链为模板，克隆OsPIP2；7。采用嵌套PCR的方法，将3×HA标签的碱基序列连接在去除终止子OsPIP2；7基因序列的3'端，然后将此核酸片段插入到植物双元表达载体pHB中，构建pHB-OsPIP2；7-3×HA载体。以中花11成熟胚愈伤组织为材料，采用农杆菌介导的方法进行遗传转化，经过在含有潮霉素的培养基上筛选和分化，获得阳性转化子。提取转基因植株的DNA，进行潮霉素阳性植株的筛选。将阳性植株进行RNA表达检测，获得OsPIP2；7基因表达量上升的转基因阳性植株。为了检测转基因植株中OsPIP2；7蛋白的表达情况，提取OsPIP2；7基因表达上升的转基因植株膜蛋白后，用HA抗体进行杂交的Western-blot检测结果显示，OsPIP2；7-HA蛋白在转基因阳性植株体内过量表达。获得的过表达OsPIP2；7转基因新材料，将为研究OsPIP2；7基因的生物学新功能提供试验基础。

关键词: 水稻, 水孔蛋白, HA标签, 蛋白表达

Abstract: To analyze the function of OsPIP2; 7 under environmental stresses, specific primers were designed to clone OsPIP2; 7 from the complementary DNA of rice variety Zhonghua 11. The nucleotide sequence of 3×HA tag was connected to 3'-end of OsPIP2; 7 with removed terminator by nested PCR amplification, and then the DNA fragments were inserted into pHB vector to construct pHB-OsPIP2; 7-3×HA. The calli of of mature embryo from Zhonghua 11 were used to conduct the genetic transformation by Agrobacterium -mediated transformation method. After screening and transformation on a culture medium containing hygromycin, the positive transformants were obtained. Western-blot results revealed that OsPIP2;7-HA was over-expressed in transgenic plant at protein level when using HA as the first antibody. This study will help the subsequent research of OsPIP2; 7 function and mechanism.

Key words: Rice, Aquaporin, HA tag, Protein expression

中图分类号:

Q785

王晓静, 孙林静, 孙玥, 张融雪, 李军玲, 闫双勇, 苏京平. HA标签的水稻水孔蛋白OsPIP2;7基因植物双元表达载体构建及转基因水稻的获得[J]. 华北农学报, 2019, 34(5): 59-64. doi: 10.7668/hbnxb.201750580.

WANG Xiaojing, SUN Linjing, SUN Yue, ZHANG Rongxue, LI Junling, YAN Shuangyong, SU Jingping. Construction of Plant Binary Expression Vector for OsPIP2; 7 with HA-tag and Its Transformation in Rice[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(5): 59-64. doi: 10.7668/hbnxb.201750580.

http://www.hbnxb.net/CN/Y2019/V34/I5/59

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HA标签的水稻水孔蛋白OsPIP2;7基因植物双元表达载体构建及转基因水稻的获得

Construction of Plant Binary Expression Vector for OsPIP2; 7 with HA-tag and Its Transformation in Rice

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HA标签的水稻水孔蛋白OsPIP2;7基因植物双元表达载体构建及转基因水稻的获得

Construction of Plant Binary Expression Vector for OsPIP2; 7 with HA-tag and Its Transformation in Rice

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